Abstract

Objectives The purpose of this study is to understand whether naturally occurring genetic variation in the promoter of chromogranin B (CHGB), a major constituent of catecholamine storage vesicles, is functional and confers risk for cardiovascular disease.

Background CHGB plays a necessary (catalytic) role in catecholamine storage vesicle biogenesis. Previously, we found that genetic variation at CHGB influenced autonomic function, with association maximal toward the 5′ region.

Methods Here we explored transcriptional mechanisms of such effects, characterizing 2 common variants in the proximal promoter, A-296C and A-261T, using transfection/cotransfection, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP). We then tested the effects of promoter variation on cardiovascular traits.

Results The A-296C disrupted a c-FOS motif, exhibiting differential mobility shifting to chromaffin cell nuclear proteins during EMSA, binding of endogenous c-FOS on ChIP, and differential response to exogenous c-FOS. The A-261T disrupted motifs for SRY and YY1, with similar consequences for EMSA, endogenous factor binding, and responses to exogenous factors. The 2-SNP CHGB promoter haplotypes had a profound (p = 3.16E-20) effect on blood pressure (BP) in the European ancestry population, with a rank order of CT<AA<<CA<AT on both systolic blood pressure (SBP) and diastolic blood pressure (DBP), accounting for ≈2.3% to ≈3.4% of SBP/DBP variance; the haplotype effects on BP in vivo paralleled those on promoter activity in cella. Site-by-site interactions at A-296C and A-261T yielded highly nonadditive effects on SBP/DBP. The CHGB haplotype effects on BP were also noted in an independent (African ancestry) sample. In normotensive twins, parallel effects were noted for a pre-hypertensive phenotype, BP response to environmental stress.

Conclusions The common CHGB promoter variants A-296C and A-261T, and their consequent haplotypes, alter binding of specific transcription factors to influence gene expression in cella as well as BP in vivo. Such variation contributes substantially to risk for human hypertension. Involvement of the sex-specific factor SRY suggests a novel mechanism for development of sexual dimorphism in BP.

The sympathoadrenal system exerts minute-to-minute control over cardiac output and vascular tone. Genes governing catecholaminergic processes may play a role in the development of hypertension (1). Sympathoadrenal catecholamine secretion is exocytotic (all or none), releasing not only catecholamines but also the acidic proteins with which catecholamines are stored. The chromogranins/secretogranins comprise a family of acidic, soluble proteins that are stored in secretory granules with different hormones, transmitters, and neuropeptides throughout the endocrine and nervous system (2). Chromogranin B (CHGB), first described in the 1980s (3–5), is a major catecholamine storage vesicle core protein and seems to play a necessary role in the biogenesis of catecholamine secretory vesicles (6).

CHGB is differentially expressed in neuroendocrine diseases, and its measurement may serve in the diagnosis and staging of such conditions (7–15). Interaction of CHGB with signaling molecules such as the inositol-1,4,5-trisphosphate-activated calcium channel (16) may influence cytosolic calcium and ultimately risk for such disease states as Alzheimer's disease, epilepsy, or schizophrenia (17). In addition, polymorphisms in the CHGB gene may be associated with schizophrenia in Chinese and Japanese populations (18,19).

Previously, we described genetic variation at the CHGB locus, and concluded that sex and CHGB interact to influence blood pressure (BP) (26). Since the association of CHGB genetic variation to BP was maximal toward the 5′ end of the gene, and such variation predicted quantitative changes in CHGB expression, we turned to potential transcriptional mechanisms. Here, we characterize the proximal promoter region of CHGB, discovering 2 common polymorphisms that disrupt transcription factor binding, giving rise to systemic hypertension. One of these sites recognizes the sex-specific factor, sex-determining region Y (SRY), providing insight into the sexual dimorphism of BP.

Methods

Subjects and clinical characterization

Subjects were volunteers, and each gave informed, written consent to protocols approved by local institutional review boards. Recruitment procedures, definitions, and confirmation of subject diagnoses are according to previous reports.

Primary care population with extremes of high and low BP

European Ancestry

As previously described (26), we ascertained 951 European-ancestry subjects, approximately half male and half female, from the highest and lowest fifth diastolic blood pressure (DBP) percentiles of a large primary care population in the Kaiser-Permanente Medical Group of Southern California (27). The DBP criterion was chosen because of the heritability of DBP (28). The statistical power of association between biallelic deoxyribonucleic acid (DNA) markers and human quantitative trait loci can be substantially augmented by the sample subjects from opposite (upper and lower) ends of the trait distribution (29–31); and analyses of the quantitative trait in extreme subjects (as opposed to dichotomization of the trait) further enhances power (32). This population sample afforded us >90% power (29,30) to detect genotype association with a trait when the genotype contributes as little as 2.5% to the total variation in males (even at p < 10−8); the power is even higher in females (31). Evaluation included physical examination, blood chemistries, hemogram, and extensive medical history questionnaire. Of the hypertensive group, 40.6% were receiving antihypertensive medications, whereas none in the normotensive group was receiving such drugs. The subjects are described in Online Table 1. Overall, 1.98% of subjects were excluded because of elevated serum creatinine (>1.5 mg/dl).

Black population with blood pressure extremes

Sub-Saharan African Ancestry

Three hundred fifty-seven adult Nigerians (approximately one-half male, one-half female) selected from the highest (n = 191) and lowest (n = 190) 25th percentiles of population BP were included as a replication sample for CHGB promoter variant effects on BP. This population has been described (33).

Twin pairs

European Ancestry

In studies of the influence of CHGB polymorphism on the pressor response to environmental stress in vivo, 156 twin pairs and 80 siblings (312 individual subjects) were evaluated. The response of BP to cold stress (by immersion of 1 hand in ice water for 1 min) was evaluated as previously described (34); responses wherein DBP increased after cold stress were analyzed. Zygosity (69% monozygotic and 31% dizygotic pairs) was confirmed by extensive microsatellite and single nucleotide polymorphism (SNP) genotyping, as described (35). Twins ranged in age from 15 to 84 years; 10% were hypertensive. Twins in these allelic/haplotype association studies were self-identified as of European (white) ancestry, to guard against potential artifactual effects of population stratification.

Statistical analyses

Haplotype blocks were visualized in Haploview (36), while haplotype assignments in individual subjects were performed by the HAP algorithm (37) in individual subjects with both A-296C and A-261T genotypes. Chi-square tests were performed to test for deviations from the Hardy-Weinberg equilibrium (HWE). When testing for associations of haplotypes with continuous/quantitative BP traits, sex, age, and body mass index (BMI) were included as covariates in the univariate tests of the general linear model using SPSS version 11.5 software (SPPS Inc., Chicago, Illinois). Both the final measured BP and that BP adjusted for the effects of antihypertensive medication (38) were analyzed. Each factor was then assessed for significance using standard analysis of variance (ANOVA) F-tests (39). Haplotype analyses were both (diploid) individual based as well as chromosome based; here, each haplotype allele (as opposed to a haplotype allele pair) was considered and analyzed separately, using the outcomes and characteristics of the subject carrying that allele (40). Associations between BP status and allele, genotype or haplotype were analyzed in n × 2 tables by either ANOVA or by SHEsis (41). Twin analyses were conducted in 2 ways: twin trait heritability (h2) was estimated in SOLAR (Southwest Foundation for Biomedical Research, San Antonio, Texas) (42); twin descriptive and inferential statistics were computed by using generalized estimating equations in SAS software (SAS Institute, Cary, North Carolina), to account for intrapair correlations (35). A p value of ≤0.05 was considered significant.

Results

Patterns of linkage disequilibrium across CHGB locus

To visualize association patterns, 16 SNPs (each in HWE) were scored and plotted by Haploview as pairwise linkage disequilibrium (LD) parameter r2 across the ∼14 kbp locus. The proximal promoter (including common variants A-296C and A-261T) was maintained within a single block in both white subjects and black subjects. The allele and genotype frequencies differed between white and black populations (Online Table 2). Although pairwise r2 values were generally higher in white subjects than in black subjects, just 2 LD blocks spanned the locus in each group (Fig. 1A). The original resequencing strategy and SNP discovery have been described (26).

Domains and motifs in the CHGB promoter

Figure 1B diagrams known motifs in the CHGB promoter, and superimposes common variants. Functional domains in the core/proximal promoter (such as the TATA box, cyclic-adenosine monophosphate response element, and G/C-rich regions) were invariant in ∼180 people (∼360 chromosomes) subjected to systematic polymorphism discovery by resequencing.

CHGB promoter variant A-296C

The A-296C variant lies in an evolutionarily conserved region among humans, nonhuman primates, and other mammals (Online Fig. 2). During EMSA, a labeled oligonucleotide representing the C allele was shifted by PC12 nuclear proteins; specificity was suggested by displacement with the same (C) allele when unlabeled (Online Fig. 3).

A-296C occurred in a potential recognition site for transcription factor c-FOS, with a 7/8 base consensus match for the A allele (43), declining to 6/8 for the C allele (Fig. 2A). When interrogated by ChIP, endogenous c-FOS binding to the motif was detected in all 4 haplotypes, although unexpectedly more intense for the A-296C than the A-296 allele (Fig. 2A).

(A) Endogenous c-FOS: motif and chromatin immunoprecipitation (ChIP). A-296 creates a 7/8 bp match with c-FOS. Experimentally, endogenous c-FOS can be captured by ChIP on all 4 2-single nucleotide polymorphism (SNP) haplotypes, although more intense at -296C than at A-296. The sequence match for the c-FOS binding site is on the reverse complement (i.e., minus) strand. The c-FOS core motif, TGASTCAC, is found in the JASPAR (43) transcription factor binding database. The IUPAC (International Union of Pure and Applied Chemistry) symbols can be found at http://www.bioinformatics.org/sms/iupac.html. (B) Exogenous c-FOS: effect of cotransfected c-FOS on transcriptional activity of chromogranin B (CHGB) promoter haplotypes in PC12 chromaffin cells. c-FOS (solid bars) consistently increases CHGB promoter expression, although the magnitude is haplotype-dependent, especially in the -261T context. Open bars indicate pcDNA3.1 (vector). ANOVA = analysis of variance.

When a plasmid expressing c-FOS was cotransfected into PC12 cells with CHGB promoter/reporters, all 4 CHGB haplotypes responded (p = 2.36E-9), although unequally (p = 7.23E-5). On a haplotypic background of the A-261 allele, c-FOS stimulated A-296 and A-296C similarly, although on a background of the -261T allele, the transcriptional response of A-296 was far greater than that of A-296C (Fig. 2B). Thus, the A-296C response to exogenous c-FOS seemed to be context dependent.

CHGB promoter variant A-261T

A-261T variant also occurs in an evolutionarily conserved region (Online Fig. 4). During EMSA, the T allele was more effectively shifted by PC12 nuclear proteins than the A allele (Online Fig. 5). During EMSA, an oligonucleotide spanning the A allele was shifted by PC12 nuclear proteins, while the T allele was shifted to a lesser degree; specificity was suggested, especially for the A allele, by displacement with the same allele when unlabeled (Online Fig. 5).

A-261T occurred in potential recognition motifs for the transcription factors SRY and YY1: the A allele displayed a superior match to both SRY (5 of 6 bases) (44,45) and YY1 (6 of 6 bases) (43) motifs, as compared with the T allele (Fig. 3B). Involvement of endogenous SRY and YY1 was probed by ChIP (Fig. 3A): for both SRY and YY1, the A allele was more effectively bound than the T allele, on either A-296C haplotypic background.

When cotransfected with a YY1 expression plasmid, CHGB promoter reporter activity increased for each haplotype (p = 7.6E-10), and the effect was more prominent for the A-261 allele than for the A-261T allele, regardless of A-296C context (p = 1.64E-4) (Fig. 3B).

CHGB promoter common variants A-296C and A-261T

Implications for Hypertension in the Population

Here we studied BP trait-extreme subjects of European ancestry, to enhance statistical power (29,30). Chromosome-based haplotype analysis on subjects dichotomized into 2 groups (higher BP vs. lower BP) indicated that subjects with the less common AT or CA haplotypes had a strong tendency to be hypertensive (odds ratio [OR]: 4.898 for AT haplotype, p = 3.19E-11; OR: 3.84 for CA haplotype, p = 3.64E-10). Subjects with the more common haplotype CT had a strong tendency to be normotensive (OR: 0.637, 95% confidence interval: 0.524 to 0.773, p = 4.64E-6). The most common haplotype AA had no effect on blood pressure status. The overall effect of CHGB haplotypes on BP status was substantial (Fig. 4A,Table 2), whether analyzed by chromosome/haplotype (global chi-square = 93.9, p = 3.16E-20), or by diploid haplotype pairs (global chi-square = 75.0, p = 4.92E-13).

Discussion

Overview

Patients with hypertension often exhibit increased sympathetic activity (47,48), and hypertension tends to develop in persons with sympathetic overactivity (49,50). Suppression of CHGB expression in chromaffin cells leads to a reduction in the number of catecholamine secretory granules, whereas ectopic expression of CHGB in nonneuroendocrine cells, which normally do not contain regulated secretory machinery, leads to granule biogenesis (6). In light of the emerging secretory biology of CHGB, we undertook the present study to probe how heredity shapes human functional responses in the sympathetic neuroeffector junction, using CHGB as a likely focal point in the pathogenesis of essential hypertension. Recently, we reported CHGB haplotype effects on BP across the CHGB locus, suggesting that the major effect was located in the 5'/promoter region (26), and the effect of CHGB on intermediate traits seems to be quantitative rather than qualitative; thus, we focused here on promoter variation at the CHGB locus. We also noted a sex-dependent effect of CHGB polymorphism on BP (26); here, we defined the effect of genetic variation on gene expression, and found evidence that the response of the gene to the male-determination factor SRY was altered by 1 promoter variant (A-261T), raising a potential mechanism for the well-known sexual dimorphism in BP.

CHGB promoter common variants

Transcriptional mechanisms of action. We identified 2 common variants in the CHGB proximal promoter: A-296C and A-261T. Here, we established that these 2 variants occur in evolutionary conserved regions, and can create or interrupt particular transcriptional control motifs. We probed these processes in 2 ways: by exposing the variants to the exogenous factors, and by testing whether the endogenous factors recognize the motifs.

A-296C Variant: c-FOS Motif

A c-FOS motif was activated by the exogenous factor and bound by the endogenous factor (Fig. 2). The c-FOS, a b-ZIP (leucine zipper) factor of the immediate/early class, may heterodimerize with a variety of other such family members (e.g., c-JUN) to trigger transcription, especially by activating AP-1 sites.

The SRY protein, testis-determining factor, is a high-mobility group box factor best known as an initiator of male development, in which its major transcriptional targets may include SOX9 (52). Of note for hypertension, we previously found that promoter A-261T exerted a sex-specific effect on population BP, with the effect confined to males (26). Here, we provide a basis for that sex-specific effect, because only males express the SRY factor, encoded by the SRY locus on human chromosome Yp11.31. While we focused on the SRY motif match in the A-261T region of the CHGB promoter (Fig. 3A), and demonstrated its binding (Fig. 3A) and trans-activation (Fig. 3B) by SRY, other members of the SOX transcription factor family, including the SRY target SOX9, may share similar consensus DNA target motifs (53) (i.e., SRY as WACAAW; SOX9 as AACAAT), and hence, both constitute potential CHGB trans-activators.

Interactions A-296C by A-261T

The likelihood of site-by-site interactions within the CHGB promoter (Fig. 4C) is suggested by 2 previous observations. First, the YY1 promoter responds transcriptionally to c-FOS (54). Second, the multifunctional factor YY1 interacts noncovalently with a variety of other transcription factors, including members of the b-ZIP family such as CREB (55). While we have documented an interaction in cis between A-296C and A-261T in transfected CHGB promoter haplotype/luciferase reporter plasmids (296-by-261 interaction, p = 3.07E-07) (Online Table 1), we have not yet explored factor interactions in trans during such transfections.

CHGB promoter common variants and hypertension across populations

The LD analysis across the CHGB locus indicated that the proximal CHGB promoter, including common variants A-296C and A-261T, is maintained within 1 block (Fig. 1A) in both white subjects and black subjects.

We enhanced power to detect genetic associations by using population trait-extreme values (29,30). We then found that haplotype effects upon BP in the population were highly significant (Fig. 4), indeed far more significant than the effects of single SNPs alone (Table 2). Substantially greater effects on BP by the A-296C/A-261T haplotypes (Fig. 4A) than by either SNP alone (Table 2), also speaks toward functional SNP-by-SNP interactions in the CHGB promoter (see transcription factor section in preceding text).

In the European ancestry population, the rank order of haplotype effects on SBP or DBP was AT>CA>>AA>CT (Fig. 4D, left), which is the same pattern of CHGB promoter haplotype activity in cella (Fig. 1C), lending weight to the viewpoint that altered CHGB transcription underlies the BP differences between haplotypic groups.

In an independent (African) population, allele and haplotype frequencies differed substantially from those in subjects of European ancestry (Online Table 2). Even so, CA haplotype copy number (0, 1, 2) influenced BP in the Nigerian sample (Fig. 4E), with the same directional effect found in white subjects (Fig. 4B). Although haplotype AT was found in substantial numbers in Nigerians (134 chromosomes), AT carriers did not display elevated BP, suggesting other factors (such as differences in environment or genetic background) influencing BP in this population, or the inclusion of other variants in the promoter LD block in subjects of African ancestry (Fig. 1A).

A previous large, genome-wide association case/control study, the WTCCC (Wellcome Trust Case Control Consortium) study, did not find association of the CHGB locus to hypertension (56). How is our study different? First of all, the WTCCC study used the Affymetrix 500K gene chip, whose average marker spacing of ∼3 × 109/500 × 103, or ∼6 kbp, did not include the CHGB promoter variants considered here; indeed, the closest CHGB promoter variant studied on the Affymetrix 500K chip in the WTCCC study was rs236129, which is 4,729 bp upstream of the cap site. Second, the WTCCC study employed single point associations, rather than studying haplotype or SNP-by-SNP interaction effects (which were crucial in our analyses). Third, because of relatively sparse marker spacing, the HapMap approach employed in the WTCCC study does not fully capture the full spectrum of potentially causal allelic variation at candidate loci (57,58). Finally, the WTCCC study used unselected/unphenotyped population controls (59); the high population prevalence (∼24%) of hypertension thus greatly diminishes the power of the WTCCC study to detect associations with hypertension. By contrast, our approach (31), using population trait (BP) extremes, offers substantially greater statistical power to detect genetic associations; indeed, we estimate that our sample has >80% power to detect loci contributing as little as ∼2.5% of BP variance.

Intermediate trait

In longitudinal studies, the pressor response to environmental (cold) stress is an effective predictor of future development of hypertension (60,61). We therefore evaluated whether CHGB genetic variation in the transcriptional control region might influence this risk predictor.

In a series of twin pairs, CHGB promoter common haplotypes influenced ΔDBP in the cold stress test (Fig. 5), with A-296/A-261 elevating and -296C/-261T diminishing the pressor response, findings that are in rank order with basal BP effects in the population (AA<CT) (Fig. 4A). Haplotypes associated with much higher BP in the population extreme subjects (AT, CA) (Fig. 4A) were too infrequent in this predominantly normotensive twin sample for meaningful conclusions to be drawn.

Conclusions

Common genetic variants in the CHGB proximal promoter seem to exert a powerful, interactive effect on BP. The CHGB promoter variants A-296C and A-261T differed substantially in transcriptional efficiency during luciferase reporter activity assays (Fig. 1C), and such activity paralleled SBP and DBP in the population (Fig. 4D, right). Particular transcription factors (c-FOS at A-296C; YY1 and SRY at A-261T) differed in activity at the variant sites (Figs. 2 and 3); such effects were captured by both cotransfection and ChIP. Differential SRY effects at A-261T suggest a mechanism that might ultimately contribute to the sexual dimorphism of BP in the population. Substantially greater effects on BP of the variants in combination (rather than as individual SNPs) suggest intrapromoter A-296C by A-261T interactions. Finally, CHGB promoter variants predict change in BP in response to environmental stress even in predominantly normotensive subjects, suggesting an early pathway by which hypertension may ultimately be mediated.

Thus, common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression, and later the early heritable responses to environmental stress, and finally resting/basal BP in the population (Fig. 6). These results point to new molecular strategies for probing autonomic control of the circulation, and ultimately the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension.

Appendix

Footnotes

This study is supported by the Department of Veterans Affairs, National Institutes of Health (NIH), the NIH/National Heart, Lung, and Blood Institute (HL58120), the NIH/National Center on Minority Health and Health Disparities-sponsored (MD000220) EXPORT/Comprehensive Research Center in Health Disparities Minority Health Center, and the NIH/National Center for Research Resources-sponsored (RR00827) General Clinical Research Center.

(2007) Identification of residues participating in the interaction between an intraluminal loop of inositol 1,4,5-trisphosphate receptor and a conserved N-terminal region of chromogranin B. Biochim Biophys Acta1774:502–509.

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